We aim to discover new classes of antimicrobial compounds that are active against methicillin/multidrug resistant Staphylococcus aureus (MRSA). Currently, MRSA infections cause more deaths each year in the U.S. than HIV/AIDS, and existing therapies are failing. We have uncovered evidence that beta lactam resistance in MRSA, which is mediated in part by the expression of a gene (mecA) encoding a resistant transpeptidase (PBP2A), requires a functional wall teichoic acid (WTA) biosynthetic pathway. Based on this, we have hypothesized that small molecules that prevent the expression of WTAs by inhibiting the first enzyme in the WTA pathway (TarO) will render MRSA susceptible to beta lactams. Consistent with this hypothesis, preliminary studies have shown that tunicamycin, a natural product that selectively inhibits TarO, indeed sensitizes MRSA to methicillin. (Unfortunately, tunicamycin has eukaryotic toxicity.) Herein, we propose a set of genetic experiments to confirm that methicillin resistance requires the expression of anionic polymer WTAs. We plan to develop and carry out a high throughput screen to identify small molecules that render MRSA strains sensitive to methicillin. This will be done by screening the compounds in duplicate in both the presence and absence of methicillin. Compounds that kill MRSA strains only in the presence of low methicillin concentrations will be examined to assess whether they inhibit WTA biosynthesis. Several techniques will be used to validate hits, including testing for the presence of WTAs through phage infection and chemical extraction. We also have a small molecule available whose antibiotic activity is antagonized by TarO inhibitors, allowing for rapid testing of the most promising hits. Confirmed non-toxic WTA inhibitors would be candidates for evaluating the utility of combination therapy for treating MRSA infections and could also be used to assess whether such inhibitors delay the development of beta lactam resistance in susceptible S. aureus strains. PUBLIC HEALTH RELEVANCE: Methicillin-resistant Staphylococcus aureus (MRSA) infections pose a large threat to public health in the United States, causing nearly 19,000 deaths in 2005. Polymers attached to the surface of S. aureus (wall teichoic acids, WTAs) are required for these bacteria to cause infections in patients. Compounds that stop WTA production could potentially serve as new therapeutics to combat MRSA.